Abstract
Fructooligosaccharide (FOS) has been used in infant formula and conventional foods as prebiotics. Short chain FOS (FOSSENCETM) is produced by a patented process of biotransformation of sucrose by the action of enzyme from live microbial cells, hence toxicology studies were initiated to assess its safety. The objective of the present study was to determine safety of FOSSENCETM in acute, 14-day, and subchronic (90-day) toxicity studies. In acute and 14-day studies, administration of the FOSSENCETM to Wistar rats did not cause any mortality or clinical signs and changes in body weights, feed consumption, and gross pathology at the doses of 2000, 5000, and 9000 mg/kg body weight. In the subchronic (90-day) toxicity study, FOSSENCETM was administered by oral gavage to Wistar rats at the doses of 0, 2000, 5000, and 9000 mg/kg/day for 90 days. No treatment-related clinical signs or mortalities were observed. Similarly, no treatment-related toxicologically or biologically significant changes in body weight, feed consumption, ophthalmological findings, neurological effects, hematology, clinical chemistry, urinalysis, and gross pathological findings were noticed. However, statistically significant increase in weight of cecum (without correlative microscopic change) was noted at all the test item-treated groups in males and females and was considered to be a trophic effect and not a toxic effect in rats.
Introduction
Fructooligosaccharide (FOS), also known as oligofructose or oligofructan, is classified as “Generally Recognized as Safe” (GRAS) under Federal Food, Drug and Cosmetic Act (FD&C Act) (US Food and Drug Administration (FDA); GRN 000605 and 000623). 1,2 FOS is composed of sucrose molecules to which one, two, or three additional fructose units are added by β-2,1-glycosidic linkages to the fructose unit of sucrose. 3 Though reported in 1980, interest in FOS increased in recent years following establishment of potential health benefits, FOS commercial use emerged in the 1980s in response to consumer demand, and in recent years, FOS has received tremendous attention for its potential health benefits. FOS occurs naturally in many plants 3,4 and as a prebiotic has been shown to exhibit beneficial health effects by stimulating the growth of bifidobacteria in the human colon. 5 –7 The health benefits derived from the colonic fermentation of FOS in humans are well-documented. 8,9 The properties of FOS include low sweetness intensity, low caloric values, and soluble dietary fiber which is noncariogenic. Additionally, FOS has been claimed to have beneficial effects such as improved mineral absorption and ability to decrease the levels of phospholipids, serum cholesterol, and triglycerides. 7,10 Further, FOS is added in many food products or as food ingredients, in addition to infant formulas because of its prebiotic effects, and also can improve food taste and texture. FOS is industrially produced from sucrose by microbial enzymes with transfructosylating activity. Most of these enzymes have been found in fungi such as Aspergillus sp., Aureobasidium sp., Arthrobacter sp., and Fusarium sp. 11 The estimated daily intake of FOS is in the range of 6.2 g/day (mean consumer) to 12.8 g/day (90th percentile consumer) in food excluding infants. The addition of FOS at levels up to 20 g/day in the conventional foods for general population and at levels up to 4.2 g/day in infant foods is safe. 2,12 FOS is also intended for use in non-exempt infant formula at the maximum intended addition levels of 400 mg FOS/100 ml in starter formula as consumed and 500 mg FOS/100 mL in follow-on-formula as consumed. In the United States, FOS is available as a nutritional supplement at recommended doses of up to 4–8 g/day to promote the growth of bifidobacteria.
FOS compositions were proposed in several GRAS determinations that received “no questions” letters from the US FDA. 12,13 In the European Union, FOS is approved for use in infant and follow-on formulas in combination with galactooligosaccharide (GOS) at levels up to 8 g (90% GOS and 10% FOS)/L. 14 The food standards of Australia and New Zealand concluded in 2013 that no adverse effects were observed in studies conducted in healthy infants at amounts of FOS up to 3.0 g/L for periods ranging from 1 week to approximately 3 months.
Various in vivo studies in animals have demonstrated that FOS is not digested by the digestive enzymes of the alimentary canal, and FOS passes unchanged into the colon where it is subsequently hydrolyzed and fermented by microflora. 5,15,16 In an acute study, Lethal Dose, 50% (LD50) was determined to be greater than 9 g/kg in Sprague–Dawley rats and in the 6-week repeat dose toxicity study, no treatment-related toxicity findings were observed up to 4.5 g/kg when FOS was administered through oral gavage. 17 In addition, a 90-day feeding study in rats up to 30% FOS (20.4 g/kg/day) revealed soft stool, diarrhea, increased intestinal weights, and cecal distention which were attributed to nondigestible nature of FOS. 12,13,18,19 There were no toxicological findings in urinalysis, hematology, clinical chemistry, gross, and histopathology. Based on the available safety related animal studies with FOS, the FDA GRAS and also the European Scientific Committee on Food and Food Standards Australia and New Zealand have described the safety nature of FOS.
FOSSENCETM (oligofructose/FOS) is a FOS containing product manufactured using a consortium of microorganisms (Aureobasidium pullulans and Pachysolen tannophilus) in a manner similar to other FOS products and its specifications are essentially equivalent with other available products that have received “no questions” letters for GRAS Determinations Notified to the US FDA. 12,13 FOSSENCETM has been produced through patented biotransformation process. To evaluate safety and establish the no observed adverse effect level (NOAEL) of FOSSENCETM, various studies (acute, 14-day dose range finding study, and subchronic 90-day toxicology) were conducted in Wistar rats.
Material and methods
Test item
FOS (FOSSENCETM) used for the experiments was provided by Tata Chemicals Limited (Tamilnadu, India) and stored at ambient conditions (+15°C to +25°C). The details of physicochemical properties of FOSSENCETM has been tabulated in Table 1.
Physicochemical properties of FOS.
Abbreviations: COA: Certificate of Analysis; FOS: fructooligosaccharide.
Compliance statement
The acute, 14-day, and subchronic (90-day) studies in Wistar rats were performed in accordance with the Organisation for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice [C(97)186/Final] and US FDA Good Laboratory Practice for Non-clinical Laboratory Studies (21 CFR Part 58). The study was conducted in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC (http://www.aaalac.org), 2001) accredited facility. The experimental projects were approved by the Institutional Animal Ethics Committee. All procedures were in compliance with the Committee for Purpose of Control and Supervision on the Experiments on Animals (CPCSEA), guidelines of India.
Acute toxicity study
The study of FOS product FOSSENCETM was performed at Eurofins Advinus Limited (Bengaluru, Karnataka, India) to determine maximum tolerable dose (MTD) after single dose administration. For these investigations, young adult healthy male and female Wistar rats (HsdHanTM) bred at Eurofins Advinus were used. The FOSSENCETM was mixed in Milli-Q water and administered as a single oral dose at the dose levels of 2000, 5000, and 9000 mg/kg (n = 5 rats/sex/group). The highest dose of 9000 mg/kg was considered as maximum feasible dose from oral administration perspective considering high viscosity factor and dose volume. Milli-Q water was administered to control group rats. The dose volume employed was 15 mL/kg body weight. Dose formulations were analyzed for FOSSENCETM contents. The rats were observed for clinical signs or mortality, and body weights and feed consumption were measured. All the rats were euthanized under isoflurane anesthesia on day 15, and gross pathological examinations were performed.
Dose range study (14-day rat study)
This 14-day dose range finding study was performed to select the dose levels for 90-day repeat dose study in rats. For these investigations, young adult healthy male and female Wistar rats (HsdHanTM) bred at Eurofins Advinus were used. The FOSSENCETM was mixed in Milli-Q water and administered through oral gavage route at the dose levels of 2000, 5000, and 9000 mg/kg/day (n = 5 rats/sex/group) for 14 consecutive days. Milli-Q water was administered to control group rats. The dose volume employed was 15 mL/kg body weight. The doses were selected based on the results of single dose toxicity study. Dose formulations were analyzed for FOSSENCETM contents from day 1 formulation samples. The rats were observed for clinical signs or mortality, and body weights and feed consumption were measured. All the rats were euthanized under isoflurane anesthesia and blood samples were collected by retro-orbital puncture on day 15 for clinical pathology (hematology, coagulation, and clinical chemistry), and organ weights and gross pathological examination were done on the same day. Based on the increased cecum weight observed in both sexes at all the doses tested, microscopic examination was performed on cecum, colon, duodenum, jejunum, and ileum from all dose group animals.
Subchronic (90-day) study
This 90-day study was performed as per OECD guidelines for testing of chemicals (Test Guideline No. 408, “Repeated Dose 90-Day Oral Toxicity Study in Rodents” adopted on September 21, 1998). For these investigations, young adult healthy male and female Wistar rats (HsdHanTM) bred at Eurofins Advinus were used. The FossenceTM was mixed in Milli-Q water and administered through oral gavage route at the dose levels of 2000, 5000, and 9000 mg/kg/day (n = 10 rats/sex/group) for 90 consecutive days. In addition, two recovery groups such as control recovery and high dose recovery were included (n = 5 rats/sex/group). Milli-Q water was administered to control group rats. The dose volume employed was 15 mL/kg body weight. The vehicle or test item solution was not administered to the recovery groups for 28 days following the 90-day dosing period. The doses were selected based on the results of 14-day repeat dose toxicity study. The dose volume was calculated for individual animals on the first day of the treatment period (day 1) and was adjusted according to the most recent body weights recorded during the treatment period. Dose formulations were analyzed for FOSSENCETM contents thrice during the treatment period, that is, day 1 and on months 2 and 3.
Animals
Wistar rats from Eurofins Advinus were examined for good health and the suitability for the study and acclimatized for 5 days before the start of the treatment. All animals were housed (two/cage) with a temperature of 20–24°C, relative humidity 65–67%, and 12 h light and 12 h dark cycle. All animals were fed ad libitum with a standard diet (Teklad Global 14% protein rodent maintenance pellet diet manufactured by Envigo Laboratories, Venray, The Netherlands) and filtered water. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object. At the commencement of the treatment, the weight variation of rats did not exceed ±20% of the mean body weight in each sex and group. These 7- to 9-week-old rats were randomly distributed to different groups by the body weight stratification method using Provantis™ software (Version 8.7.3, Instem LSS, Staffordshire ST15OSD, United Kingdom).
Parameters evaluated
Clinical signs, body weights, and feed consumption. All rats were observed once daily for changes in appearance, behavior, clinical/toxic signs, and neurological changes and twice daily for morbidity and mortality. Detailed clinical examination was done prior to the test article administration on day 1 and at weekly intervals thereafter during the treatment period. Individual body weight and feed consumption were recorded prior to test article administration on day 1 and at weekly intervals thereafter till the end of the experiment period. Fasting body weight was recorded prior to termination.
Ophthalmological examination and functional observation battery. Ophthalmological examination was performed with an ophthalmoscope (direct ophthalmoscope, New York, USA; WelchAllyn) prior to start of treatment, end of the treatment period, and at the end of the recovery period. The neurobehavioral examinations such as home cage observations, handling observations, open-field observations, sensory observations, neuromuscular observations, and physiological observation (rectal temperature) were conducted during 13th week of the treatment period for all the rats in main toxicity groups and during last week of the recovery period for the rats in recovery groups.
Clinical pathology. All rats were fasted overnight before blood collection on day 91 for main toxicity groups and on day 119 for recovery groups. The blood was collected for all groups by retro-orbital plexus puncture with the help of a fine capillary tube under isoflurane (Abbott Laboratories, Illinois, USA) anesthesia. The hematological, coagulation, and clinical chemistry parameters listed in Table 2 were determined using the ADVIA 2120 hematology system (Bayer Health Care LLC, Tarrytown, New York, USA), Start-4 coagulation analyzer (Diagnostica stago, 92600 Asnieres, France), and Dimension RxL Max clinical Chemistry System (Dade Behring Inc., Newark, Delaware, USA), respectively.
Parameters evaluated in hematology, coagulation, and clinical chemistry.
a Differential Leukocyte parameters and their respective abbreviations are as follows: neutrophils (Neut), lymphocytes (Lymp), monocytes (Mono), eosinophils (Eosi), and basophils (Baso).
b Measured using refractometry method (Refractometer—digital handheld “Pocket” urine specific gravity refractometer by (PAL-10 S, ATAGO Co., Ltd 32-10) Honcho, Itabashi, Tokyo, Japan).
c Analyzed using Multistix 10 SG strips, read with Clinitek status analyzer (Bayer Healthcare LLC, UK).
d Recorded manually.
Necropsy, organ weights, and histopathology. At the end of the treatment (day 91) and at the end of the recovery period (day 119), all rats were fasted overnight (approximately 16 h). On the day of necropsy, all rats were weighed (fasting body weight), exsanguinated under isoflurane anesthesia, and subjected to detailed necropsy. The gross pathological changes, if any, were recorded for each rat. Necropsy observation included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs, and tissues. The organs and tissues listed in Table 3 from all rats were collected and fixed using 10% Neutral Buffered Formalin (monosodium phosphate dihydrate, disodium hydrogen phosphate, and formaldehyde, all from Rankem, Gurugram, Haryana, India). The organs marked with X were weighed. The paired organs were weighed together and combined weight was presented. The organ weight ratios as percentage of body and brain weight were calculated based on the fasted body weight and brain weight.
Organ/tissue collected, preservated, and examined.
a Bone marrow smears were prepared from femur marrow and stained using Giemsa stain.
b Weighed with and without content.
c Eyes were collected in Davidson’s fluid (isopropyl alcohol and glacial acetic acid were from Rankem, India and Spectrochem, India, respectively)
d Decalcified prior to sectioning.
e Inflated with 10% neutral-buffered formalin before fixation.
f Weighed after formalin fixation.
g Prostate + seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands.
Histopathological examination was carried out on all the preserved organs and tissues of vehicle control (G1) and 9000 mg FOS/kg/day group rats. In addition, all gross lesions from all the animals were examined microscopically. There were no test item-related changes observed at 9000 mg/kg/day dose group and hence, histopathological evaluation was not carried out for lower dose (2000 mg FOS/kg/day and 5000 mg FOS/kg/day) groups and recovery groups. The tissues were processed for routine paraffin embedding and 4–5 µm sections were stained with Mayer’s hematoxylin and eosin stain.
Statistical analysis
The individual data were subjected to statistical analyses. The analyzed data were expressed as mean ± SD. All quantitative variables like body weight, feed consumption, laboratory investigation (hematology, coagulation, and clinical chemistry), and organ weight data were tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) within the group before performing one-way analysis of variance (ANOVA) modeling by treatment groups. When the data were found to be nonoptimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and control group was done using Dunnet’s test when the overall treatment ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5% (p < 0.05) level. The neurological observations (neuromuscular observation/body temperature/body weights) was calculated and analyzed by SYSTAT Statistical package Version 12.0 using one way ANOVA and t test.
Results
Stability and analysis of the test item in vehicle
High Performance Liquid Chromatography (HPLC) method using refractive index detection was validated at Eurofins Advinus. Validated HPLC method was used for the stability evaluation and concentration verification of prepared dose formulation samples. The stability of the test item in the vehicle at 65 and 650 mg/mL concentrations showed that the test item was found to be stable for 24 h at room temperature and 4 days at refrigerated temperature. The dose formulations were analyzed in single dose, dose range 14-day, and subchronic (90-day) study. The dose formulation results of 90-day study (day 1) are shown in Table 4. The results of dose formulation analysis indicated that the analyzed concentrations were found to be within ±10% of the claimed concentration and the relative standard deviation (%RSD) was less than 10% of the claimed concentration for FOS set (GF2, GF3, and GF4). For other sugars (Glycerol, Arabitol, Fructose, Glucose, and sucrose), the analyzed concentrations were within ±20% of the claimed concentration and the %RSD was less than 15% of the claimed concentration. The control group did not show any peaks for test item contamination.
Concentration data of FOSSENCETM in dose formulation samples of day 1 samples of the 90-day study.a
RSD: relative standard deviation; FOS: fructooligosaccharide; SD: standard deviation.
a Acceptance criteria: Formulations are considered acceptable if mean results are within ± 10% of the nominal concentration and the RSD is equal to or less than 10% for FOS set (GF2, GF3, and GF4) and within ±20% of the nominal concentration, and the RSD is equal to or less than 15% for other sugars (glycerol, arabitol, fructose, glucose, and sucrose).
Acute toxicity study
In the single dose toxicity study, oral gavage administration of FOSSENCETM in Wistar rats did not reveal any clinical signs, mortality, on body weight, and feed consumption (data not shown) at 2000, 5000, and 9000 mg/kg body weight. Necropsy at the end of study (day 15 post-dose) did not reveal any gross pathological abnormalities. Hence, the MTD was considered to be more than 9000 mg/kg body weight. Similar findings were reported in acute oral toxicity study, and LD50 for oral administration of FOS to rats was more than 9000 mg/kg body weight. 20
Dose range study (14-day rat study)
No clinical signs or mortality were observed at the tested dose levels of 2000, 5000, and 9000 mg/kg/day. The body weights were unaffected at all the doses tested. The slight decrease in feed consumption observed during treatment days 4–8 and 8–11 at 9000 mg/kg/day, however food consumption during days 11–14 was comparable to the control group. Hence slight decrease in food consumption that was observed during initial days of the treatment was considered as transient non-adverse finding. There were no test item-related changes observed in hematology, coagulation, and clinical chemistry parameters as well as in organs weight and gross pathology. There were no test item-related microscopic changes observed in cecum, colon, duodenum, jejunum, and ileum of both the sexes. Fourteen-day repeat dose oral gavage administration of FOSSENCETM to Wistar rats did not cause any adverse toxicological changes on the evaluated parameters at 2000, 5000, and 9000 mg/kg/day doses.
Subchronic (90-day) rat study
Mortality, clinical signs, and ophthalmological examination
There were no deaths, relevant clinical signs, or abnormal ophthalmological findings noticed at any of the dose levels in this study. Few clinical signs such as sparse hair loss/local alopecia in four rats (control recovery male—1, high dose male—1, and high dose recovery female—2) and lacrimation in one rat (high dose female) were observed during the experimental period. The clinical sign of sparse hair loss/local alopecia was observed randomly in 4/100 rats and hence considered as incidental findings. The clinical sign of lacrimation (slight in nature) was also considered to be an incidental finding due to its isolated occurrence.
Functional observation battery
There were no treatment-related changes observed in neurological/functional examination carried out at the end of treatment period for the main toxicity treatment groups and at the end of recovery period for the toxicity recovery groups.
Body weights
Body weight data are presented in Table 5, and growth curves are presented in Figures 1 and 2. Body weights were unaffected at 2000 and 5000 mg/kg/day doses in males and at all the doses tested in females as compared to the control group. The statistically significant lower body weights were observed on day 90 (p < 0.05) at 9000 mg/kg/day when compared to the control group in males. The body weights were slightly lower (without statistical significance) at 9000 mg/kg/day in both main and recovery group males for the most part of the treatment period from week 7 till the end of the treatment period and considered partially reversible at the end of the recovery period.
Mean body weights (g) values.
a Significantly lower than the vehicle control group at p < 0.05.
Growth curves of male in terms of mean body weight (g). G1/G1 R, G2, G3, and G4/G4 R represents 0, 2000, 5000, and 9000 mg FOS/kg/day, respectively. FOS: fructooligosaccharide.
Growth curves of female in terms of mean body weight (g). G1/G1 R, G2, G3, and G4/G4 R represents 0, 2000, 5000, and 9000 mg FOS/kg/day, respectively. FOS: fructooligosaccharide.
Feed consumption
Feed consumption data are presented in Table 6 and Figures 3 and 4. The food consumption was unaffected at 2000 mg/kg/day (G2) in males and females as compared to the control group. The statistical significant changes (decrease) in feed consumption were observed at the doses of 5000 and 9000 mg/kg/day (p < 0.05) in males and females during the treatment period and considered reversible during the recovery period.
Mean feed consumption (g/rat/day) values.
a Significantly lower than the vehicle control group at p < 0.05.
b Significantly lower than the vehicle control/ vehicle control recovery group at p < 0.05.
Feed consumption curves of male in terms of mean feed consumption (g/rat/day). G1/G1 R, G2, G3, and G4/G4 R represents 0, 2000, 5000, and 9000 mg FOS/kg/day, respectively. FOS: fructooligosaccharide.
Feed consumption curves of female in terms of mean feed consumption (g/rat/day). G1/G1 R, G2, G3, and G4/G4 R represents 0, 2000, 5000, and 9000 mg FOS/kg/day, respectively. FOS: fructooligosaccharide.
Clinical pathology
Hematology and coagulation
Hematology and coagulation data are presented in Table 7. There were no test item-related biologically significant adverse effects observed in hematological and coagulation parameters of both the sexes across the groups. There were few statistically significant differences in hematology parameters in FOS-treated animals compared to controls included decreased hemoglobin at 5000 mg/kg/day in males; decreased mean corpuscular hemoglobin concentration at all FOS-treated groups in males and at 5000 and 9000 (main and recovery) mg/kg/day in females; increased mean platelet volume at 2000 and 5000 mg/kg/day in males and 9000 mg/kg/day recovery in males and females; decreased absolute eosinophils at 9000 mg/kg/day in males; and decreased reticulocytes (both absolute and %) at 9000 mg/kg/day recovery females. In the coagulation parameters, decreased prothrombin time values at 9000 mg/kg/day recovery males were noted.
Mean hematology and coagulation values.
Hct: hematocrit; Hgb: hemoglobin; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; MCV: mean corpuscular volume; MPV: mean platelet volume; Plat: platelets; RBC: red blood cells; WBC: white blood cells; DLC: differential leukocyte counts; Rectic: reticulocytes; Neut: Neutrophils; Lymp: lymphocytes; Mono: monocytes; Eosi: eosinophils; Baso: basophils; PT: prothrombin time; APTT: activated partial thromboplastin time.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/ vehicle control recovery group at p < 0.05.
Clinical chemistry and urinalysis
Mean values of clinical chemistry data are presented in Table 8. There were no test item-related biologically significant adverse effects observed in clinical chemistry parameters of both the sexes across the groups. There are occasional sporadic findings of statistically significant differences in the following parameters from FOS-treated rats compared to controls included decreased total cholesterol, total proteins, and globulin at 5000 and 9000 mg/kg/day in males and at 9000 mg/kg/day in females; alanine aminotransferase at 9000 mg/kg/day recovery in males; decreased potassium at 9000 mg/kg/day recovery in males; and increased alkaline phosphatase at 9000 mg/kg/day in females. There were no test item-related changes in the urinalysis parameters in treated rats compared to controls.
Values of clinical chemistry.
ALT: alanine aminotransferase; Alb: albumin; ALP: alkaline phosphatase; A/G: albumin/globulin ratio; AST: aspartate amino transferase; GGT: gamma-glutamyl transpeptidase; BUN: blood urea nitrogen; Creat: creatinine; CK: creatine kinase; Ca: calcium; Cl: chloride; Glob: globulin; Glu: glucose; Pi: inorganic phosphorous; K: potassium; Na: sodium; T.Chol: total cholesterol; T.Pro: total plasma protein; T.Bil: total bilirubin; Trig: triglycerides.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/ vehicle control recovery group at p < 0.05.
Organ weights and gross pathology
Organ weights (absolute and relative) data are presented in Tables 9 to 14. Increase in absolute and relative cecum weight (with and without content) was observed at 9000 mg/kg/day in both the sexes. However, this change was not associated with any microscopic changes and hence considered as test item-related non-adverse effect. The cecum weight change was completely reversed in the recovery males, whereas in females, it was partially recovered.
Organ weight males.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/vehicle control recovery group at p < 0.05.
Organ to body weight ratios males.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/vehicle control recovery group at p < 0.05.
Organ to brain weight ratios males.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/vehicle control recovery group at p < 0.05.
Organ weight females.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/vehicle control recovery group at p < 0.05.
Organ to body weight ratios females.
a Significantly higher/lower than the vehicle control group at p < 0.05.
b Significantly higher/lower than the vehicle control/vehicle control recovery group at p < 0.05.
Organ to brain weight ratios females.
a Significantly higher/lower than the vehicle control group at p < 0.05.
Similar increase in cecum weight was also present at 2000 and 5000 mg/kg/day in both the sexes and was attributed to test item administration.
All other statistically significant differences observed in organ weight and their ratios were considered incidental as the changes were minimal in magnitude and/or lacked the microscopic correlation.
There were no test item-related gross changes observed in male and female rats.
Histopathology
Histopathology results from control and high dose (9000 mg/kg/day) groups have been listed in Table 15. There were no test item-related microscopic changes observed in male and female rats at all the doses tested. All the microscopic findings observed in males and females at 9000 mg/kg/day dose were considered incidental/spontaneous and not related to test item administration, as they were distributed randomly across the groups and/or normally present in rats of this age. In addition, observed microscopic findings were comparable to vehicle control group.
Incidence of selected histopathological findings.
Discussion
This subchronic study was conducted to assess the systemic toxicity potential of the FOSSENCETM when administered orally by gavage to Wistar rats for a period of 90 consecutive days and to determine the reversibility of effects following 28 days recovery period. The doses employed in this study were 0, 2000, 5000, and 9000 mg/kg/day.
There were no deaths, relevant clinical signs, or abnormal ophthalmological findings reported at all the dose levels in this study. Functional observational battery tests revealed no test item-related effects on home cage, handling, open-field, sensory, neuromuscular, and physiological observations up to the highest dose of 9000 mg/kg/day. Body weights were unaffected by the treatment at 2000 and 5000 mg/kg/day doses in males and at all the doses tested in females. The feed consumption was unaffected by the treatment at 2000 mg/kg/day throughout the treatment period. The changes observed in body weights in males at 9000 mg/kg/day and feed consumption in both sexes at 5000 and 9000 mg/kg/day were considered as minimal changes. All rats were found normal without any test item-related effects on clinical signs during the in-life phase of the experimental period, and there were no patho-physiological or test item-related microscopic lesions in all the terminally killed rats. In addition, the changes observed in body weights were partially reversible and feed consumption was completely reversible by the end of 28-day recovery period. Hence, the minimal changes observed in body weights and food consumption was considered as non-adverse findings.
There were no test item-related adverse changes reported in hematological, coagulation, clinical chemistry, and urine parameters in both sexes in all test item-treated groups, either at the end of the treatment period or at the end of recovery period. All the statistically significant changes observed were considered incidental and toxicologically insignificant as the alterations were of minimal in magnitude and/or lacked the dose progression and also microscopic correlation. Decreased absolute (73%) and relative (74%) reticulocytes’ counts at 9000 mg/kg/day recovery females was considered as incidental finding as similar changes was not observed during treatment period. In addition, males were not affected and also no changes were observed in red blood cells and hemoglobin.
The mean absolute and relative cecum weight to body weight and brain weight (with or without content) were significantly increased at 9000 mg/kg/day in both the sexes and were attributed to test item administration. Similar increase in cecum weight was also present at 2000 and 5000 mg/kg/day in both the sexes and was attributed to test item administration. The change observed in cecum weight was found to be completely reversible in males, whereas in females, it was found to be partially reversible. At necropsy, no test item-related gross pathological changes were observed at all dose levels. There were no test item-related histopathological changes observed in all the evaluated organs and tissues including cecum. As no microscopic lesions observed in cecum at the highest dose of 9000 mg/kg/day, the increased cecal weight was considered test item-related non-adverse finding. Cecal enlargement is a common finding in toxicology studies of FOS products. Similar findings such as, reduced feed consumption in both sexes, lower body weights in males, dose-related increase in absolute and relative cecal weights of both sexes without any associated histopathology, and no test item findings in hematology, clinical chemistry, and urinalysis were reported when oligofructose was administered at the dietary concentrations 4.96% and 9.91%. 21 Genta et al. 22 reported that dietary concentration of 0, 188, or 3760 mg oligofructose/kg/day (equivalent to 0, 340, or 6800 mg/kg, yacon root containing oligofructose) for 4 months in Wistar rats resulted in increased absolute and relative weights of the Gastrointestinal Tract (GI) due to enlargement of the cecum; however, no effects on body weights and feed intake were seen.
Cecum is an area of significant bacterial fermentation, with increased amounts of short chain fatty acids that are produced by bacterial fermentation after large amounts of non-adsorbed carbohydrate and dietary fiber enter the cecum and colon. 23 –25 Tokunaga et al. 26 reported similar findings wherein FOS administration at the dietary concentration of 0%, 10%, and 20% produced dose-dependent significant increases in the weights of the cecum and colon in 6-week rat study. It has also been reported that increased cecal weight and/or mucosal hypertrophy in rodents represents a physiological adaptation to increased osmotic forces when high doses of undigestible substances are consumed; the effect is reversible after test item is withdrawn from the diet. 27 –29 The large doses of FOS may result in cecal enlargement indicative of higher cecum weight, which was considered to be a trophic effect and not a toxic effect. 12
In conclusion, the oral gavage administration of FOSSENCETM up to 9000 mg FOS/kg body weight/day is considered safe in Wistar rats without any adverse toxicological findings when administered for 90 consecutive days. The “No Observed Adverse Effect Level (NOAEL)” established was 9000 mg FOS/kg body weight/day in this study under the test conditions employed.
Footnotes
Authors’ note
HK, DPS, GSR, SKV, and SS are employees of Eurofins Advinus Limited, Bengaluru, India, where the study was performed. MJ, MG, AD, and SN are employees of Tata Chemicals Limited.
Acknowledgement
Authors are thankful to the management of Tata Chemicals Limited and Eurofins Advinus Limited.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Tata Chemicals Limited—Innovation Centre, Survey No. 315, Hissa No. 1-14, Ambedveth (V), Paud Road, Mulshi, Pune 412111, Maharashtra, India, sponsored this project.